The major goal of this application is to elucidate the function of alpha2- macroglobulin (alpha2M) as a cytokine-carrier. Specific Aim 1 focuses on the biochemistry of alpha2M-cytokine interactions. Equilibrium dissociation constants and dissociation rate constants for physiologically significant alpha2M-cytokine complexes will be determined. We will test the hypothesis that alpha2M cytokine-carrier activity and the resulting regulation of nitric oxide synthesis accounts for the endotoxin-insensitive phenotype of alpha2M gene knock-out mice. We will also study the interaction of alpha2M with macrophage deactivating cytokines to probe the mechanisms by which alpha2M regulates macrophage phenotype. In Specific Aim 2, work underway will be continued to identify recombinant polypeptides, corresponding to fragments of the alpha2M structure, that bind transforming growth factor-beta2 (TGF-beta2) and related cytokines. Identified polypeptides will be evaluated in biological systems by exogenous addition or by transfection of cells with expression constructs. In Specific Aim 3, mechanisms by which alpha2M regulates cytokine activity will be elucidated. The ability of activated alpha2M to target TGF-beta to cells that express the alpha2M receptor (LRP) will be evaluated using cultures of LRP(+/+) fibroblasts and lRP(-/-) fibroblasts, derived by gene knock-out technology. We will tests the hypothesis that native alpha2M regulates cytokine activity be altering the rate of delivery of cytokines to signalling receptors. In addition, we will determine whether high- capacity non-signalling receptors counteract the activity of alpha2M in the regulation of cytokine activity. To accomplish this last objective, endothelial cells will be transfected with an expression construct for the TGF-beta receptor, betaglycan. The goal of Specific Aim 4 is to test the hypothesis that alpha2M regulates macrophage autocrine TGF-beta activity and thus promotes macrophage activation for tumor cell killing. Macrophage activation parameters, such as nitric oxide synthesis and TNF-alpha expression, will be assessed in RAW 264.7 macrophages transfected with a TGF-beta antisense construct; in cells treated with exogenous alpha2M; and in cells transfected with constructs that drive expression of wild-type and mutant alpha2Ms. These studies will enhance our understanding of the role of alpha2M in various pathophysiologic states and specifically in cancer.